Project 4 Summary Development of a safe and efficacious vaccine that prevents HIV infection is still an unmet need. We have demonstrated that a Cytomegalovirus (CMV)-vectored vaccine expressing SIV antigens elicits high frequency, broadly distributed, effector-memory, SIV-specific CD4+ and CD8+ T cell responses that mediate complete (?control and clear?) protection of 50-60% of vaccinated RM against repeated mucosal challenge with highly pathogenic SIVmac239. Recent studies have demonstrated that only RhCMV vectors lacking the RhCMV homologs of the HCMV UL128 and UL130 genes are able to mediate this remarkable efficacy, despite similar immunogenicity of the UL128/UL130-intact vectors in terms of magnitude, distribution and functional differentiation. We further determined that the UL128/UL130-deletion had a major effect on the epitope targeting of the RhCMV vector-elicited CD8+ T cells: whereas the CD8+ T cells elicited by UL128/UL130-intact RhCMV vectors are conventionally MHC-Ia-restricted, the CD8+ T cells elicited by UL128/UL130-deleted RhCMV vectors are entirely restricted by either MHC-II or non-classical MHC-E. We have also determined that the MHC-II- and MHC-E-restricted components of the protective immune responses are independently regulated, and thus separable. Since a protective HCMV/HIV vectored vaccine will almost certainly need to induce unconventional T cell responses in people in order to mediate protection, effective clinical translation of our pre-clinical findings will depend upon the identification of the protective component of the unconventional responses (Project 1), and the delineation of the virologic mechanisms by which RhCMV elicits these efficacious responses (Projects 2-4). We have determined that UL128 and UL130 contribute to unconventional response programming by at least 2 different mechanisms: an effect on vector tropism mediated by the contribution of both UL128 and UL130 to the pentameric receptor complex (PRC) and a PRC-independent mechanism by which either UL128 or UL130 can inhibit MHC-E-restricted CD8+ T cell response priming in myeloid-derived cells. The effect of tropism on CMV vector response programming is addressed in Project 2. In Project 4 we first seek to identify the specific motifs in the UL128 and UL130 proteins which block MHC-E- restricted CD8+ T cell priming with the goal of mutating these regions to abrogate their MHC-E-response inhibition while retaining PRC function. Since MHC-E-restricted response priming is myeloid cell-dependent, whereas MHC-II responses are disfavored by vector infection of myeloid cells, this should result in a vector that preferentially elicits MHC-E-restricted responses. Second, since HCMV lacking UL128 and UL130 fails to elicit unconventional T cell responses in RM, its possible that HCMV encodes other genes that block unconventional CD8+ T cell responses. Therefore, utilizing a deletion strategy we will ascertain whether any non-essential HCMV genes have this activity. Finally, utilizing the data from Projects 2-4, Project 4 will construct the 2nd generation ?response and safety? optimized HCMV/HIV vector for manufacture and clinical testing in Project 5.